XB-ART-42850Dev Cell. January 18, 2011; 20 (1): 19-32.
Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA--miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs--in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.
PubMed ID: 21238922
PMC ID: PMC3361082
Article link: Dev Cell.
Grant support: Wellcome Trust , 090868 Wellcome Trust , 090868 Wellcome Trust , Wellcome Trust , WT090868 Wellcome Trust , Wellcome Trust , 090868 Wellcome Trust , Wellcome Trust
Genes referenced: cdknx fgf8 hes1 mdm2 myt1 neurod1 rpl8 sox3 tp53 tubb2b wnt1 zic1
Morpholinos referenced: hes1 MO1
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|Figure 1. miR-9 Expression Differs along the AP Axis(A) Whole-mount in situ hybridization for miR-9 expression in X. tropicalis using LNA probe.(B) Expression of miR-9 primary transcripts at stage 30—dorsal view. Dashed line indicates the plane of sectioning in (D). MHB is indicated with an asterisk. Scale bar, 200 μm.(C) Schematic representation of the different regions in the neural tube (red, forebrain; green, midbrain; blue, hindbrain) and transverse sections from the forebrain and hindbrain (red, progenitors; purple, intermediate zone; blue, neurons).(D) In situ hybridization for miR-9 precursors in transverse sections from stage 30 embryos. CNS tissue is outlined with a dashed line. Scale bar, 20 μm.(E) FISH for miR-9a-1 (in red) combined with immunohistochemistry (IHC) for Sox3 (marker for neural progenitors) in stage 30 embryo. CNS tissue is outlined with a dashed line. DNA is stained with DAPI (4,6-diamidino-2-phenylindole). Scale bar, 20 μm.|
|Figure 5. miR-9 Regulates the Expression of hairy1 In Vivo(A) Sequence alignment of the predicted miR-9 binding site in HES1 homologs in human, mouse, Xenopus, and zebrafish. Positions that have a single, fully conserved residue are marked with an asterisk. Seed-complementary region is boxed in red.(B) HeLa cells were transfected with WT Xenopus hairy1 (xhairy1_WT), mouse Hes1 (mHes1), or mutant hairy1 (xHairy1_Mut) reporter together with either scrambled (Control) or miR-9 precursors (miR-9). Luciferase expression was normalized and expressed relative to the control levels. Error bars represent SD.(C) Design of target protector morpholino (Hairy1 TP) directed against hairy1 miR-9 binding site. Seed region is boxed in red.(D) Hairy1 TP alleviates the repression of hairy1 luciferase reporter when cotransfected with miR-9 precursors but has no effect on the repression of other miR-9 targets. Error bars represent SD.(E) In situ hybridization for miR-9 (miR-9a-1 transcript) and hairy1 in stage 30 embryos. Shown are whole mounts and transverse sections through the respective brain areas.(F) Double-fluorescent in situ for hairy1 (red) and miR-9a-1 (green) shows mutually exclusive pattern of expression along the AP axis.(G) miR-9 MO and hairy1 TP lead to expansion of the hairy1-positive domain (red arrowheads) along the AP axis, as shown by in situ hybridization.(H) Quantification of the change in hairy1 mRNA expression using qRT-PCR.(I) Hes1 mRNA levels in N1E neuroblastoma cells are downregulated when miR-9 is overexpressed and increased when it is knocked down using LNA inhibitors. In all graphs, data are presented as mean values, and error bars represent SEM.|
|Figure 7. Mechanism of miR-9 Function(A) Forebrain sections of miR-9 MO or hairy1 TP-injected embryos analyzed for CyclinD1, p27Xic1, and Fgf8 expression by in situ hybridization.(B) Hindbrain sections of miR-9 MO or hairy1 TP-injected embryos analyzed for CyclinD1, p27Xic1, Wnt1, and Zic1 expression.(C) Representative western blot for endogenous p53 protein levels in forebrain or hindbrain tissue isolated from X. tropicalis embryos injected with control MO, miR-9 MO, or hairy1 TP. Numbers represent the mean from three experiments, 60 embryos each.(D) Real-time PCR analysis for Mdm2 expression, normalized for the ribosomal protein RPL8 (n = 3 experiments, 20 embryos each). Error bars represent SEM.(E) Model for miR-9 function in cell survival and progenitor proliferation.|